Salinosporamides and methods for use thereof

ABSTRACT

The present invention is based on the discovery that certain fermentation products of the marine actinomycete strains CNB392 and CNB476 are effective inhibitors of hyperproliferative mammalian cells. The CNB392 and CNB476 strains lie within the family Micromonosporaceae, and the generic epithet  Salinospora  has been proposed for this obligate marine group. The reaction products produced by this strain are classified as salinosporamides, and are particularly advantageous in treating neoplastic disorders due to their low molecular weight, low IC 50  values, high pharmaceutical potency, and selectivity for cancer cells over fungi.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/147,622 filed Jun. 7, 2005, now issued as U.S. Pat. No.7,176,233; which is divisional application of U.S. application Ser. No.10/838,157 filed Apr. 30, 2004, now issued as U.S. Pat. No. 7,176,232;which is a continuation-in-part application of U.S. application Ser. No.10/600,854 filed Jun. 20, 2003, now issued as U.S. Pat. No. 7,179,834;which claims the benefit under 35 U.S.C. § 119(e) to U.S. applicationSer. No. 60/391,314 filed Jun. 24, 2002. The disclosure of each of theprior applications is considered part of and is incorporated byreference in the disclosure of this application.

GRANT INFORMATION

This invention was made in part with government support under Grant No.CA44848 awarded by the National Institutes of Health, National CancerInstitute. The United States government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to anti-neoplastic agents, and moreparticularly to salinosporamides and their use as anti-neoplasticagents.

2. Background Information

Neoplastic diseases, characterized by the proliferation of cells notsubject to the normal control of cell growth, are a major cause of deathin humans. Clinical experience in chemotherapy has demonstrated that newand more effective cytotoxic drugs are desirable to treat thesediseases. Indeed, the use of anti-neoplastic agents has increased due tothe identification of new neoplasms and cancer cell types withmetastases to different areas, and due to the effectiveness ofantineoplastic treatment protocols as a primary and adjunctive medicaltreatment for cancer.

Since anti-neoplastic agents are cytotoxic (poisonous to cells) they notonly interfere with the growth of tumor cells, but those of normalcells. Anti-neoplastic agents have more of an effect on tumor cells thannormal cells because of their rapid growth. Thus, normal tissue cellsthat are affected by anti-neoplastic agents are rapidly dividing cells,such as bone marrow (seen in low blood counts), hair follicles (seen byway of hair loss) and the GI mucosal epithelium (accounting for nausea,vomiting, loss of appetite, diarrhea). In general, anti-neoplasticagents have the lowest therapeutic indices of any class of drugs used inhumans and hence produce significant and potentially life-threateningtoxicities. Certain commonly-used anti-neoplastic agents have unique andacute toxicities for specific tissues. For example, the vinca alkaloidspossess significant toxicity for nervous tissues, while adriamycin hasspecific toxicity for heart tissue and bleomycin has for lung tissue.

Thus, there is a continuing need for anti-neoplastic agents that areeffective in inhibiting the proliferation of hyperproliferative cellswhile also exhibiting IC₅₀ values lower than those values found forcurrent anti-neoplastic agents, thereby resulting in marked decrease inpotentially serious side effects.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that certainfermentation products of the marine actinomycete strains CNB392 andCNB476 are effective inhibitors of hyperproliferative mammalian cells.The CNB392 and CNB476 strains lie within the family Micromonosporaceae,and the generic epithet Salinospora has been proposed for this obligatemarine group. The reaction products produced by this strain areclassified as salinosporamides, and are particularly advantageous intreating neoplastic disorders due to their low molecular weight, lowIC₅₀ values, high pharmaceutical potency, and selectivity for cancercells over fungi.

In one embodiment of the invention, there is provided compounds havingthe structure (I):

wherein:

-   -   R₁ to R₃ are each independently —H, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclic, substituted heterocyclic, cycloalkyl, substituted        cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted        thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H,        acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;    -   Each R₄ is independently alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, cycloalkyl, substituted cycloalkyl;    -   E₁ to E₄ are each independently —O, —NR₅, or —S, wherein R₅ is        —H or C₁-C₆ alkyl; and    -   x is 0 to 8.

In a further embodiment of the invention, there are provided compoundshaving the structure (II):

wherein:

-   -   R₁ to R₃ are each independently —H, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclic, substituted heterocyclic, cycloalkyl, substituted        cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted        thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H,        acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;    -   Each R₄ is independently alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, cycloalkyl, substituted cycloalkyl;    -   E₁ to E₄ are each independently —O, —NR₅, or —S, wherein R₅ is        —H or C₁-C₆ alkyl; and    -   x is 0 to 8.

In another embodiment of the invention, there are provided compoundshaving the structure (III):

wherein:

-   -   R₁ to R₃ are each independently —H, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclic, substituted heterocyclic, cycloalkyl, substituted        cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted        thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H,        acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl,    -   each R₄ is independently alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, cycloalkyl, substituted cycloalkyl,    -   E₁ to E₄ are each independently —O, —NR₅, or —S, wherein R₅ is        —H or C₁-C₆ alkyl, and    -   x is 0 to 8.

In still a further embodiment of the invention, there are providedcompounds having the structure (IV):

In a further embodiment of the invention, there are provided compoundshaving the structure (V):

In a further embodiment of the invention, there are provided compoundshaving the structure (VI):

In another embodiment, there are provided pharmaceutical compositionsincluding at least one compound of structures I-VI in a pharmaceuticallyacceptable carrier therefor.

In another embodiment, there are provided articles of manufactureincluding packaging material and a pharmaceutical composition containedwithin the packaging material, wherein the packaging material includes alabel which indicates that the pharmaceutical composition can be usedfor treatment of cell proliferative disorders and wherein thepharmaceutical composition includes at least one compound of structuresI-VI.

In yet another embodiment, there are provided methods for treating amammalian cell proliferative disorder. Such a method can be performedfor example, by administering to a subject in need thereof atherapeutically effective amount of a compound having structures I-VI.

In an additional embodiment, there are provided methods for producing acompound of structures I-VI having the ability to inhibit theproliferation of hyperproliferative mammalian cells. Such a method canbe performed, for example, by cultivating a culture of a Salinospora sp.strains CNB392 or CNB476 (ATCC # PTA-5275, deposited on Jun. 20, 2003,pursuant to the Budapest Treaty on the International Deposit ofMicroorganisms for the Purposes of Patent Procedure with the PatentCulture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A.) and isolating from theculture at least one compound of structure I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of an exemplary compound of theinvention, Salinosporamide A, with relative stereochemistry.

FIG. 2 depicts a phylogenetic tree illustrating the phylogeny of“Salinospora”.

FIG. 3 depicts the chemical structure of Etoposide, an anti-neoplasticagent in therapy against several human cancers.

FIG. 4 compares the cytotoxic activity and dose response curves ofSalinosporamide A and Etoposide.

FIG. 5 is a block diagram depicting an exemplary separation scheme usedto isolate Salinosporamide A.

FIGS. 6-14 set forth NMR, IR, and UV spectroscopic data used toelucidate the structure of Salinosporamide A.

FIG. 15 sets forth the signature nucleotides that strains CNB392 andCNB476 possess within their 16S rDNA, which separate these strainsphylogenetically from all other family members of the familyMicromonosporaceae.

FIG. 16 depicts the chemical structure of an exemplary compound of theinvention, salinosporamide A (structure V), with absolutestereochemistry.

FIG. 17 ORTEP plot of the final X-ray structure of salinosporamide A,depicting the absolute stereochemistry.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, there are provided compounds having the structure(I):

wherein:

-   -   R₁ to R₃ are each independently —H, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclic, substituted heterocyclic, cycloalkyl, substituted        cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted        thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H,        acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;    -   Each R₄ is independently alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, cycloalkyl, substituted cycloalkyl;    -   E₁ to E₄ are each independently —O, —NR₅, or —S, wherein R₅ is        —H or C₁-C₆ alkyl; and    -   x is 0 to 8.

In a further embodiment of the invention, there are provided compoundshaving the structure (II):

wherein:

-   -   R₁ to R₃ are each independently —H, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclic, substituted heterocyclic, cycloalkyl, substituted        cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted        thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H,        acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;    -   Each R₄ is independently alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, cycloalkyl, substituted cycloalkyl;    -   E₁ to E₄ are each independently —O, —NR₅, or —S, wherein R₅ is        —H or C₁-C₆ alkyl; and    -   x is 0 to 8.

In one embodiment, there are provided compounds having the structure(III):

wherein:

-   -   R₁ to R₃ are each independently —H, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclic, substituted heterocyclic, cycloalkyl, substituted        cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted        thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H,        acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl,    -   each R₄ is independently alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, cycloalkyl, substituted cycloalkyl,    -   E₁ to E₄ are each independently —O, —NR₅, or —S, wherein R₅ is        —H or C₁-C₆alkyl, and    -   x is 0 to 8.

In still a further embodiment of the invention, there are providedcompounds having the structure (IV):

In a further embodiment of the invention, there are provided compoundshaving the structure (V):

In a further embodiment of the invention, there are provided compoundshaving the structure (VI):

As used herein, the term “alkyl” refers to a monovalent straight orbranched chain hydrocarbon group having from one to about 12 carbonatoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

As used herein, “substituted alkyl” refers to alkyl groups furtherbearing one or more substituents selected from hydroxy, alkoxy,mercapto, cycloalkyl, substituted cycloalkyl, heterocyclic, substitutedheterocyclic, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, aryloxy, substituted aryloxy, halogen, cyano, nitro, amino,amido, —C(O)H, acyl, oxyacyl, carboxyl, sulfonyl, sulfonamide, sulfuryl,and the like.

As used herein, “lower alkyl” refers to alkyl groups having from 1 toabout 6 carbon atoms.

As used herein, “alkenyl” refers to straight or branched chainhydrocarbyl groups having one or more carbon-carbon double bonds, andhaving in the range of about 2 up to 12 carbon atoms, and “substitutedalkenyl” refers to alkenyl groups further bearing one or moresubstituents as set forth above.

As used herein, “alkynyl” refers to straight or branched chainhydrocarbyl groups having at least one carbon-carbon triple bond, andhaving in the range of about 2 up to 12 carbon atoms, and “substitutedalkynyl” refers to alkynyl groups further bearing one or moresubstituents as set forth above.

As used herein, “aryl” refers to aromatic groups having in the range of6 up to 14 carbon atoms and “substituted aryl” refers to aryl groupsfurther bearing one or more substituents as set forth above.

As used herein, “heteroaryl” refers to aromatic rings containing one ormore heteroatoms (e.g., N, O, S, or the like) as part of the ringstructure, and having in the range of 3 up to 14 carbon atoms and“substituted heteroaryl” refers to heteroaryl groups further bearing oneor more substituents as set forth above.

As used herein, “alkoxy” refers to the moiety —O-alkyl-, wherein alkylis as defined above, and “substituted alkoxy” refers to alkoxyl groupsfurther bearing one or more substituents as set forth above.

As used herein, “thioalkyl” refers to the moiety —S-alkyl-, whereinalkyl is as defined above, and “substituted thioalkyl” refers tothioalkyl groups further bearing one or more substituents as set forthabove.

As used herein, “cycloalkyl” refers to ring-containing alkyl groupscontaining in the range of about 3 up to 8 carbon atoms, and“substituted cycloalkyl” refers to cycloalkyl groups further bearing oneor more substituents as set forth above.

As used herein, “heterocyclic”, refers to cyclic (i.e., ring-containing)groups containing one or more heteroatoms (e.g., N, O, S, or the like)as part of the ring structure, and having in the range of 3 up to 14carbon atoms and “substituted heterocyclic” refers to heterocyclicgroups further bearing one or more substituents as set forth above.

In certain embodiments, there are provided compounds of structures I-IIIwherein E₁, E₃, and E₄ are —O, and E₂ is —NH.

In certain embodiments, there are provided compounds of structures I-IIIwherein R₁ and R₂ are —H, alkyl, or substituted alkyl, and R₃ is hydroxyor alkoxy. In some embodiments, R₁ is substituted alkyl. Exemplarysubstituted alkyls contemplated for use include halogenated alkyls, suchas for example chlorinated alkyls.

The compounds of the invention may be formulated into pharmaceuticalcompositions as natural or salt forms. Pharmaceutically acceptablenon-toxic salts include the base addition salts (formed with freecarboxyl or other anionic groups) which may be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like.Such salts may also be formed as acid addition salts with any freecationic groups and will generally be formed with inorganic acids suchas, for example, hydrochloric, sulfuric, or phosphoric acids, or organicacids such as acetic, p-toluenesulfonic, methanesulfonic acid, oxalic,tartaric, mandelic, and the like. Salts of the invention include aminesalts formed by the protonation of an amino group with inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like. Salts of the invention also includeamine salts formed by the protonation of an amino group with suitableorganic acids, such as p-toluenesulfonic acid, acetic acid, and thelike. Additional excipients which are contemplated for use in thepractice of the present invention are those available to those ofordinary skill in the art, for example, those found in the United StatesPharmacopeia Vol. XXII and National Formulary Vol. XVII, U.S.Pharmacopeia Convention, Inc., Rockville, Md. (1989), the relevantcontents of which is incorporated herein by reference.

The compounds according to this invention may contain one or moreasymmetric carbon atoms and thus occur as racemates and racemicmixtures, single enantiomers, diastereomeric mixtures and individualdiastereomers. The term “stereoisomer” refers to chemical compoundswhich differ from each other only in the way that the different groupsin the molecules are oriented in space. Stereoisomers have the samemolecular weight, chemical composition, and constitution as another, butwith the atoms grouped differently. That is, certain identical chemicalmoieties are at different orientations in space and, therefore, whenpure, have the ability to rotate the plane of polarized light. However,some pure stereoisomers may have an optical rotation that is so slightthat it is undetectable with present instrumentation. All such isomericforms of these compounds are expressly included in the presentinvention.

Each stereogenic carbon may be of R or S configuration. Although thespecific compounds exemplified in this application may be depicted in aparticular configuration, compounds having either the oppositestereochemistry at any given chiral center or mixtures thereof are alsoenvisioned. When chiral centers are found in the derivatives of thisinvention, it is to be understood that this invention encompasses allpossible stereoisomers. The terms “optically pure compound” or“optically pure isomer” refers to a single stereoisomer of a chiralcompound regardless of the configuration of the compound.

Exemplary invention compounds of structure I are shown below:

Salinosporamide A exhibits a molecular structure having a variety offunctional groups (lactone, alkylhalide, amide, hydroxide) that can bechemically modified to produce synthetic derivatives. Accordingly,exemplary invention compound Salinosporamide A provides an excellentlead structure for the development of synthetic and semisyntheticderivatives. Indeed, Salinosporamide A can be derivatized to improvepharmacokinetic and pharmacodynamic properties, which facilitateadministration and increase utility of the derivatives asanti-neoplastic agents. Procedures for chemically modifying inventionsalinosporamide compounds to produce additional compounds within thescope of the present invention are available to those of ordinary skillin the art.

Salinosporamide A shows strong cytotoxic activity against human coloncancer cells in the HTC-116 cell assays. The IC₅₀ of 11 ng/mL exceedsthe activity of etoposide (see FIG. 3, IC₅₀ 828 ng/mL), an anticancerdrug used for treatment of a number of cancers, by almost two orders ofmagnitude (see FIG. 4). This high activity makes inventionsalinosporamides excellent candidates for use in the treatment ofvarious human cancers, especially slow growing, refractile cancers forwhich there are no therapies. Salinosporamide A is specific toinhibition of mammalian cells and shows little anifungal activityagainst Candida albicans (IC₅₀ 250 μg/mL) and no antibacterial activity(Staphylococcus aureus, Enterococcus faecium). The IC₅₀ ofSalinosporamide A is far lower than the strongest chemotherapeuticagents currently in use or in clinical trials.

Salinosporamide A is a fermentation product of the marine actinomycetestrains CNB392 and CNB476. These strains are members of the orderActinomycetales, which are high G+C gram positive bacteria. The noveltyof CNB392 and CNB476 is at the genus level. Invention compounds setforth herein are produced by certain “Salinospora” sp. In someembodiments, invention compounds are produced by “Salinospora” sp.strains CNB392 and CNB476. To that end, the CNB476 strains of“Salinospora” sp. were deposited on Jun. 20, 2003, pursuant to theBudapest Treaty on the International Deposit of Microorganisms for thePurposes of Patent Procedure with the Patent Culture Depository of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 U.S.A. under ATCC Accession No PTA-5275.

As is the case with other organisms, the characteristics of“Salinospora” sp. are subject to variation. For example, recombinants,variants, or mutants of the specified strain may be obtained bytreatment with various known physical and chemical mutagens, such asultraviolet ray, X-rays, gamma rays, andN-methyl-N′-nitro-N-nitrosoguanidine. All natural and induced variants,mutants, and recombinants of the specified strain which retain thecharacteristic of producing a compound of the invention are intended tobe within the scope of the claimed invention.

Invention compounds can be prepared, for example, by bacterialfermentation, which generates the compounds in sufficient amounts forpharmaceutical drug development and for clinical trials. In someembodiments, invention compounds are produced by fermentation of theactinomycete strains CNB392 and CNB476 in AlBfe+C or CKA-liquid media.Essential trace elements which are necessary for the growth anddevelopment of the culture should also be included in the culturemedium. Such trace elements commonly occur as impurities in otherconstituents of the medium in amounts sufficient to meet the growthrequirements of the organisms. It may be desirable to add small amounts(i.e. 0.2 mL/L) of an antifoam agent such as polypropylene glycol (M.W.about 2000) to large scale cultivation media if foaming becomes aproblem. The organic metabolites are isolated by adsorption onto anamberlite XAD-16 resin. For example, Salinosporamide A is isolated byelution of the XAD-16 resin with methanol:dichlormethane 1:1, whichaffords about 105 mg crude extract per liter of culture. SalinosporamideA is then isolated from the crude extract by reversed-phase flashchromatography followed by reverse-phase HPLC and normal phase HPLC,which yields 6.7 mg of Salinosporamide A. FIG. 5 sets forth a blockdiagram outlining isolation and separation protocols for inventioncompounds.

The structure of Salinosporamide A was elucidated by a variety of NMRtechniques, mass spectroscopy, IR, and UV spectroscopy, as set forth inFIGS. 6-14.

The absolute structure of salinosporamide A, and confirmation of theoverall structure of salinosporamide A, was achieved by single-crystalX-ray diffraction analysis (see Example 3).

The present invention also provides articles of manufacture includingpackaging material and a pharmaceutical composition contained within thepackaging material, wherein the packaging material comprises a labelwhich indicates that the pharmaceutical composition can be used fortreatment of disorders and wherein the pharmaceutical compositionincludes a compound according to the present invention. Thus, in oneaspect, the invention provides a pharmaceutical composition including acompound of the invention, wherein the compound is present in aconcentration effective to treat cell proliferative disorders. Theconcentration can be determined by one of skill in the art according tostandard treatment regimen or as determined by an in vivo animal assay,for example.

Pharmaceutical compositions employed as a component of inventionarticles of manufacture can be used in the form of a solid, a solution,an emulsion, a dispersion, a micelle, a liposome, and the like, whereinthe resulting composition contains one or more invention compounds as anactive ingredient, in admixture with an organic or inorganic carrier orexcipient suitable for enteral or parenteral applications. Compoundsemployed for use as a component of invention articles of manufacture maybe combined, for example, with the usual non-toxic, pharmaceuticallyacceptable carriers for tablets, pellets, capsules, suppositories,solutions, emulsions, suspensions, and any other form suitable for use.The carriers which can be used include glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, medium chainlength triglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening and coloring agents andperfumes may be used.

The compositions of the present invention may contain other therapeuticagents as described below, and may be formulated, for example, byemploying conventional solid or liquid vehicles or diluents, as well aspharmaceutical additives of a type appropriate to the mode of desiredadministration (for example, excipients, binders, preservatives,stabilizers, flavors, etc.) according to techniques such as those wellknown in the art of pharmaceutical formulation.

Invention pharmaceutical compositions may be administered by anysuitable means, for example, orally, such as in the form of tablets,capsules, granules or powders; sublingually; buccally; parenterally,such as by subcutaneous, intravenous, intramuscular, or intracisternalinjection or infusion techniques (e.g., as sterile injectable aqueous ornon-aqueous solutions or suspensions); nasally such as by inhalationspray; topically, such as in the form of a cream or ointment; orrectally such as in the form of suppositories; in dosage unitformulations containing non-toxic, pharmaceutically acceptable vehiclesor diluents. Invention compounds may, for example, be administered in aform suitable for immediate release or extended release. Immediaterelease or extended release may be achieved by the use of suitablepharmaceutical compositions comprising invention compounds, or,particularly in the case of extended release, by the use of devices suchas subcutaneous implants or osmotic pumps. Invention compounds may alsobe administered liposomally.

The invention further provides methods for using inventionsalinosporamide compounds of structures (I)-(VI) to inhibit theproliferation of mammalian cells by contacting these cells with aninvention salinosporamide compound in an amount sufficient to inhibitthe proliferation of the mammalian cell. One embodiment is a method toinhibit the proliferation of hyperproliferative mammalian cells. Forpurposes of this invention, “hyperproliferative mammalian cells” aremammalian cells which are not subject to the characteristic limitationsof growth, e.g., programmed cell death (apoptosis). A further preferredembodiment is when the mammalian cell is human. The invention furtherprovides contacting the mammalian cell with at least one inventionsalinosporamide compound and at least one additional anti-neoplasticagent.

In another embodiment, there are provided methods for treating amammalian cell proliferative disorder, comprising administering to asubject in need thereof a therapeutically effective amount of a compoundof structures (I)-(VI). Cell proliferative disorders that can beeffectively treated by the methods of the invention include disorderscharacterized by the formation of neoplasms. As such, inventioncompounds are anti-neoplastic agents. As used herein, “neoplastic”pertains to a neoplasm, which is an abnormal growth, such growthoccurring because of a proliferation of cells not subject to the usuallimitations of growth. As used herein, “anti-neoplastic agent” is anycompound, composition, admixture, co-mixture or blend which inhibits,eliminates, retards or reverses the neoplastic phenotype of a cell. Incertain embodiments, the neoplasms are selected from mammory, small-celllung, non-small-cell lung, colorectal, leukemia, melanoma, pancreaticadenocarcinoma, central nervous system (CNS), ovarian, prostate, sarcomaof soft tissue or bone, head and neck, gastric which includes thyroidand non-Hodgkin's disease, stomach, myeloma, bladder, renal,neuroendocrine which includes thyroid and non-Hodgkin's disease andHodgkin's disease neoplasms. In one embodiment, the neoplasms arecolorectal.

Chemotherapy, surgery, radiation therapy, therapy with biologic responsemodifiers, and immunotherapy are currently used in the treatment ofcancer. Each mode of therapy has specific indications which are known tothose of ordinary skill in the art, and one or all may be employed in anattempt to achieve total destruction of neoplastic cells. Chemotherapyutilizing one or more invention salinosporamide compounds is provided bythe present invention. Moreover, combination chemotherapy, chemotherapyutilizing invention salinosporamide compounds in combination with otherneoplastic agents, is also provided by the invention as combinationtherapy is generally more effective than the use of singleanti-neoplastic agents. Thus, a further aspect of the present inventionprovides compositions containing a therapeutically effective amount ofat least one invention salinosporamide compound in combination with atleast one other anti-neoplastic agent. Such compositions can also beprovided together with physiologically tolerable liquid, gel or solidcarriers, diluents, adjuvants and excipients. Such carriers, diluents,adjuvants and excipients may be found in the United States PharmacopeiaVol. XXII and National Formulary Vol XVII, U.S. Pharmacopeia Convention,Inc., Rockville, Md. (1989), the contents of which are hereinincorporated by reference. Additional modes of treatment are provided inAHFS Drug Information, 1993 ed. by the American Hospital FormularyService, pp. 522-660, the contents of which are herein incorporated byreference.

Anti-neoplastic agents which may be utilized in combination with aninvention salinosporamide compound include those provided in The MerckIndex, 11th ed. Merck & Co., Inc. (1989) pp. Ther 16-17, the contents ofwhich are hereby incorporated by reference. In a further embodiment ofthe invention, anti-neoplastic agents may be antimetabolites which mayinclude, but are not limited to, methotrexate, 5-fluorouracil,6-mercaptopurine, cytosine arabinoside, hydroxyurea, and2-chlorodeoxyadenosine. In another embodiment of the present invention,the anti-neoplastic agents contemplated are alkylating agents which mayinclude, but are not limited to, cyclophosphamide, melphalan, busulfan,paraplatin, chlorambucil, and nitrogen mustard. In a further embodimentof the invention, the antineoplastic agents are plant alkaloids whichmay include, but are not limited to, vincristine, vinblastine, taxol,and etoposide. In a further embodiment of the invention, theanti-neoplastic agents contemplated are antibiotics which may include,but are not limited to, doxorubicin (adriamycin), daunorubicin,mitomycin c, and bleomycin. In a further embodiment of the invention,the anti-neoplastic agents contemplated are hormones which may include,but are not limited to, calusterone, diomostavolone, propionate,epitiostanol, mepitiostane, testolactone, tamoxifen, polyestradiolphosphate, megesterol acetate, flutamide, nilutamide, and trilotane. Ina further embodiment of the invention, the anti-neoplastic agentscontemplated include enzymes which may include, but are not limited to,L-Asparaginase or aminoacridine derivatives which may include, but arenot limited to, amsacrine. Additional anti-neoplastic agents includethose provided in Skeel, Roland T., “Antineoplastic Drugs and BiologicResponse Modifier: Classification, Use and Toxicity of Clinically UsefulAgents,” Handbook of Cancer Chemotherapy (3rd ed.), Little Brown & Co.(1991), the contents of which are herein incorporated by reference.

In addition to primates, such as humans, a variety of other mammals canbe treated according to the method of the present invention. Forinstance, mammals including, but not limited to, cows, sheep, goats,horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine,canine, feline, rodent or murine species can be treated.

The term “therapeutically effective amount” means the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, animal or human that is being sought by theresearcher, veterinarian, medical doctor or other clinician, e.g.,lessening of the effects/symptoms of cell proliferative disorders.

By “pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The terms “administration of” and or “administering a” compound shouldbe understood to mean providing a compound of the invention to theindividual in need of treatment. Administration of the inventioncompounds can be prior to, simultaneously with, or after administrationof another therapeutic agent or other anti-neoplastic agent.

The pharmaceutical compositions for the administration of the compoundsof this invention may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general, the pharmaceutical compositions are prepared byuniformly and intimately bringing the active ingredient into associationwith a liquid carrier or a finely divided solid carrier or both, andthen, if necessary, shaping the product into the desired formulation. Inthe pharmaceutical composition the active object compound is included inan amount sufficient to produce the desired effect upon the process orcondition of diseases.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compounds of the present invention may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compounds of the present invention are employed.

Compounds and compositions of the invention can be administered tomammals for veterinary use, such as for domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual hosts. Ordinarily, dosageswill range from about 0.001 to 1000 μg/kg, more usually 0.01 to 10μg/kg, of the host body weight. Alternatively, dosages within theseranges can be administered by constant infusion over an extended periodof time, usually exceeding 24 hours, until the desired therapeuticbenefits have been obtained. It will be understood, however, that thespecific dose level and frequency of dosage for any particular patientmay be varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the hostundergoing therapy.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES

Methods and Materials

HPLC-Purification of invention compounds was accomplished by RP-MPLC onC18-solid phase (Aldrich) using a step gradient on Kontes Flex-columns(15×7 mm). Semipreparative HPLC was performed on an isocratic HPLCsystem with a Waters pump 6000H on normal phase column Si-Dynamas-60 Å(250×5 mm) or reversed phase column C18-Dynamax-60 Å, flow 2 mL/minute,with a differential refractomeric detector Waters R401.

LC-MS-The LC-MS chromatography was performed on a Hewlett-Packard systemseries HP1100 with DAD and MSD1100 detection. The separation wasaccomplished on reversed phase C18 (Agilent Hypersil ODS 5 μm, columndimension 4.6×100 mm), flow rate 0.7 mL/minute using a standardgradient: 10% acetonitrile, 15 minutes; 98% acetonitrile (Burdick &Jackson high purity solvents). The MS-detection was in ESI positivemode, capillary voltage 3500 eV, fragmentation voltage 70 eV, mass rangem/z 100-1000. The APCI-mode was measured at a flow rate of 0.5mL/minute, positive detection, capillary voltage 3000 eV, fragmentationvoltage 70 eV.

NMR-NMR spectra were measured on a Varian 300 MHz gradient fieldspectrometer with inverse-mode for ¹H or 2D-NMR spectra. The 13C andDEPT spectra were measured on a Varian 400 MHz, broad band instrument.The reference is set on the internal standard tetramethylsilane (TMS,0.00 ppm).

MS-EI-Low resolution MS-EI spectra were performed on a Hewlett-Packardmass spectrometer with magnetic sector field device, heating rate 20°C./minute up to 320° C., direct injection inlet.

FTMS-MALDI—High resolution MS data were obtained by MALDI operating modeon an IonSpec Ultima FT Mass Spectrometer.

IR-Infrared spectra were measured on a Perkin-Elmer FT infraredspectrophotometer using NaCl windows.

Example 1 Isolation and Characterization of “Salinsospora” Species,Culture Nos. CNB392 and CNB476

CNB392 and CNB476 possess signature nucleotides within their 16S rDNAwhich separate these strains phylogenetically from all other members ofthe family Micromonosporaceae (see FIG. 15) These signature nucleotideshave been determined to be a definitive marker for members of this groupwhich also have a physiological growth requirement of sodium. Signaturenucleotides were aligned to E. coli positions 27-1492 using all existingmembers of the Micromonosporaceae in the Ribosomal Database Project asof 1-31-01. For the “Salinospora” clade, 45 partially sequencedmorphotypes displayed all the signature nucleotides from positions207-468. The seven “Salinospora” isolates sequenced almost in theirentirety (see FIG. 2) displayed all of the signatures in FIG. 15.

The strains CNB392 and CNB476 form bright orange to black colonies onagar and lacks aerial mycelia. Dark brown and bright orange diffusiblepigments are produced depending upon cellular growth stage. Sporesblacken the colony surface and are borne on substrate mycelia.Vegetative mycelia are finely branched and do not fragment. Spores areproduced singly or in clusters. Neither sporangia nor spore motility hasbeen observed for these strains. CNB392 and CNB476 have an obligategrowth requirement for sodium and will not grow on typical media usedfor maintenance of other generic members of the Micromonosporaceae.CNB392 and CNB476 have been found to grow optimally on solid media TCGor M1 at 30° C.

TCG 3 grams tryptone M1 10 grams starch 5 grams casitone 4 grams yeastextract 4 grams glucose 2 grams peptone 18 grams agar (optional) 18grams agar (optional) 1 liter filtered seawater 1 liter filteredseawaterFermentaion

CNB392 and CNB476 are cultured in shaken AlBfe+C or CKA-liquid media, 1liter at 35° C. for 9 days. After 4 days 20 grams Amberlite XAD-16 resin(Sigma, nonionic polymeric adsorbent) is added.

A1Bfe + C 10 grams starch CKA 5 grams starch 4 grams yeast extract 4 mLhydrosolubles 2 grams peptone (50%) 1 gram CaCO₃ 2 grams menhaden meal 5mL KBr (aqueous 2 grams kelp powder solution, 20 2 grams chitosangrams/liter) 1 liter filtered 5 mL Fe₂(SO₄)₃ × seawater 4H₂O (8grams/liter) 1 liter filtered seawaterExtraction

The XAD-16 resin is filtered and the organic extract is eluted with 1liter ethylacetate followed by 1 liter methanol. The filtrate is thenextracted with ethylacetate (3×200 mL). The crude extract from the XADadsorption is 105 mg. Cytotoxicity on the human colon cancer cellHCT-116 assay is IC50<0.076 μg/mL.

Isolation of Salinosporamide A from CNB392

The crude extract was flash-chromatographed over C18 reversed phase (RP)using a step gradient (FIG. 5). The HCT-116 assay resulted in two activefractions, CNB392-5 and CNB392-6. The combined active fractions (51.7mg), HCT-116<0.076 μg/mL) were then chromatographed on an isocraticRP-HPLC, using 85% methanol at 2 mL/minute flow as eluent and usingrefractive index detection. The active fraction CNB392-5/6 (7.6 mg,HCT-116<0.076 μg/mL) was purified on an isocratic normal phase HPLC onsilica gel with ethyl acetate:isooctane (9:1) at 2 mL/minute.Salinosporamide A (FIG. 1) was isolated as a colorless, amorphous solidin 6.7 mg per 1 liter yield (6.4%). TLC on silica gel(dichloromethane:methanol 9:1) shows Salinosporamide A at r_(f)=0.6, noUV extinction or fluorescence at 256 nm, yellow with H₂SO₄/ethanol, darkred-brown with Godin reagent (vanillin/H₂SO₄/HClO₄). Salinosporamide Ais soluble in CHCl₃, methanol, and other polar solvents like DMSO,acetone, acetonitrile, benzene, pyridine, N,N-dimethyformamide, and thelike. ¹H NMR: (d₅-pyridine, 300 MHz), 1.37/1.66 (2H, m, CH₂), 1.70.2.29(2H, m, CH₂), 1.91 (2H, broad, CH₂), 2.07 (3H, s, CH₃), 2.32/2.48 (2H,ddd, ³J=7.0 Hz, CH₂), 2.85 (1H, broad, m, CH), 3.17 (1H, dd, ³J=10 Hz,CH), 4.01/4.13 (2H, m, CH₂), 4.25 (1H, d, ³J=9.0 Hz, CH), 4.98 (1H,broad, OH), 5.88, (1H, ddd, ³J=10 Hz, CH), 6.41 (1H, broad d, ³J=10 Hz,CH), 10.62 (1H, s, NH).

¹³C NMR/DEPT: (d₅-pyridine, 400 MHz), 176.4 (COOR), 169.0 (CONH), 128.8(═CH), 128.4 (═CH), 86.1 (C_(q)), 80.2 (C_(q)), 70.9 (CH), 46.2 (CH),43.2 (CH₂), 39.2 (CH), 29.0 (CH₂), 26.5 (CH₂), 25.3 (CH₂), 21.7 (CH₂),20.0 (CH₃).

LC-MS (ESI), t_(r)=10.0 minutes, flow 0.7 mL/minute.

m/z: (M+H)⁺ 314, (M+Na)⁺ 336; fragments: (M+H—CO₂)⁺ 292, (M+H—CO₂—H₂O)⁺270, 252, 204. Cl pattern: (M+H, 100%)⁺ 314, (M+H, 30%)⁺ 316.

-   -   LC MS (APCI): t_(r)=11.7 minutes, flow 0.5 mL/minute.        -   m/z: (M+H)⁺ 314, fragments: (M+H—CO₂—H₂O)⁺ 270, 252, 232,            216, 160.        -   Cl pattern: (M+H, 100%)⁺ 314, (M+H, 30%)⁺ 316.

EI: m/z: 269, 251, 235, 217, 204, 188 (100%), 160, 152, 138, 126, 110,81.

FTMS-MALDI: m/z: (M+H)⁺ 314.1144.

FT-IR: (cm⁻¹) 2920, 2344, s, 1819 m, 1702 s, 1255, 1085 s, 1020 s, 797s.

Molecular formula: C₁₅H₂₀ClNO₄.

Example 2 Bioactivity Assays

Salinosporamide A shows strong activity against human colon cancer cellswith an IC₅₀ of 0.011 μg/mL (see FIG. 4). The screening on antibacterialor antifungal activity shows no significant activity, see Table 1.

TABLE 1 IC₅₀ of Salinosporamide A, Assay (μg/mL) HCT-116 0.011 Candidaalbicans 250 Candida albicans (amphoterocin B resistant) NSA*Staphylococcus aureus (methecillin resistant) NSA* Enterococcus faecium(vanomycin resistant) NSA* *NSA = no significant activity

Example 3 Determination of Absolute Stereochemistry

Crystallization of a compound of structure I from ethylacetate/iso-octane resulted in single, cubic crystals, which diffractedas a monoclinic system P2(1). The unusual high unit-cell volume of 3009Å hosted four independent molecules in which different conformationalpositions were observed for the flexible chloroethyl substituent. Theassignment of the absolute structure from the diffraction anisotropy ofthe chlorine substituent resolved the absolute stereochemistry ofsalinosporamide A as 2R, 3S, 4R, 5S, 6S (FIGS. 16 and 17) with a Flackparameter of 0.01 and an esd of 0.03.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method of treating a mammalian cell proliferative disorder,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a compound having the structure:

thereby treating a mammalian cell proliferative disorder, wherein theproliferative disorder is selected from the group consisting of mammaryneoplasm, non-small-cell lung neoplasm, leukemia neoplasm, melanomaneoplasm, pancreatic adenocarcinoma neoplasm, central nervous systemneoplasm, ovarian neoplasm, prostate neoplasm, myeloma neoplasm,non-Hodgkin's neoplasm, and Hodgkin's neoplasm.
 2. The method of claim1, wherein the mammalian cell is human.
 3. The method of claim 1,wherein the proliferative disorder is a mammary neoplasm.
 4. The methodof claim 1, wherein the proliferative disorder is a non-small-cell lungneoplasm.
 5. The method of claim 1, wherein the proliferative disorderis a leukemia neoplasm.
 6. The method of claim 1, wherein theproliferative disorder is a melanoma neoplasm.
 7. The method of claim 1,wherein the proliferative disorder is a pancreatic adenocarcinomaneoplasm.
 8. The method of claim 1, wherein the proliferative disorderis a central nervous system neoplasm.
 9. The method of claim 1, whereinthe proliferative disorder is an ovarian neoplasm.
 10. The method ofclaim 1, wherein the proliferative disorder is a prostate neoplasm. 11.The method of claim 1, wherein the proliferative disorder is a myelomaneoplasm.
 12. The method of claim 1, wherein the proliferative disorderis a non-Hodgkin's neoplasm.
 13. The method of claim 1, wherein theproliferative disorder is a Hodgkin's neoplasm.
 14. The method of claim1, wherein the compound has the structure:


15. The method of claim 14, wherein the proliferative disorder is amammary neoplasm.
 16. The method of claim 14, wherein the proliferativedisorder is a non-small-cell lung neoplasm.
 17. The method of claim 14,wherein the proliferative disorder is a leukemia neoplasm.
 18. Themethod of claim 14, wherein the proliferative disorder is a melanomaneoplasm.
 19. The method of claim 14, wherein the proliferative disorderis a pancreatic adenocarcinoma neoplasm.
 20. The method of claim 14,wherein the proliferative disorder is a central nervous system neoplasm.21. The method of claim 14, wherein the proliferative disorder is anovarian neoplasm.
 22. The method of claim 14, wherein the proliferativedisorder is a prostate neoplasm.
 23. The method of claim 14, wherein theproliferative disorder is a myeloma neoplasm.
 24. The method of claim14, wherein the proliferative disorder is a non-Hodgkin's neoplasm. 25.The method of claim 14, wherein the proliferative disorder is aHodgkin's neoplasm.
 26. A method of treating cancer, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound having the structure:

wherein the cancer is selected from the group consisting of mammary,non-small-cell lung, leukemia, melanoma, pancreatic adenocarcinoma,central nervous system neoplasm, ovarian, prostate, myeloma,non-Hodgkin's cancer and Hodgkin's cancer.
 27. The method of claim 26,wherein the compound has the structure: